Tailoring the properties of (catalytically)-active inclusion bodies

Abstract

BackgroundImmobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically.ResultsWe here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein.ConclusionWe here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1081-5) contains supplementary material, which is available to authorized users.

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

“…Since CatIBs are produced heterologously in bacteria, it is not surprising that different parameters, like fusion protein design, expression conditions, and downstream processing, strongly influence not only the general success of immobilization as CatIBs but also their properties. The latter observation also has direct consequences for biocatalytic application of CatIBs as shown recently in several studies (Jäger et al 2019a;Kloss et al 2018a).…”

“…This was for example demonstrated for the lysine decarboxylase from E. coli, where the N-terminus is buried within the decameric structure of the enzyme, while the C-terminus is located at the protein surface. In accordance, the activity of the CatIBs derived from Cterminal fusion of TDoT was about six orders of magnitude higher than for the corresponding N-terminal fusion (Jäger et al 2019a;Kloss et al 2018b). Thus, in conclusion, the fusion site (N-vs C-terminal) should be carefully evaluated and if no structures are available, both sites need to be tested.…”

“…This suggests that CatIB formation not only is driven by the aggregation-inducing tag but, at least to a certain extent, also depends on the interactions of the target proteins (and/or the tag) caused by the physical proximity of the target molecules themselves. This is illustrated by the observation that the CatIB formation efficiency observed for TDoT-CatIBs of mCherry was much reduced as compared with the corresponding YFP TDoT-CatIBs (Jäger et al 2019a), which might be related to the fact that monomeric mCherry virtually lacks any hydrophobic surface patches compared with dimeric YFP ( Fig. 3a; compare mCherry: 2H5Q; eYFP: 1YFP).…”

“…It is important to note that with the same genetic construct CatIBs, mostly soluble proteins or conventional, inactive IBs can be produced by changing a single cultivation parameter (Lamm et al 2020). Therefore, it might be necessary to screen the expression conditions for potential new CatIB constructs within a certain process window, e.g., by profiling expression temperature, inductor concentration, and induction time, as simulation of protein folding and aggregation with fusion proteins is at present not feasible (Krauss et al 2017;Lamm et al 2020;Jäger et al 2019a;Huber et al 2009).…”

“…Due to their purity, consisting predominately of the aggregating target protein, they have traditionally been used for refolding studies, in which they served as an easy to separate source of pure target protein (Singh et al 2015). This long-held misconception has been challenged in recent years as more and more studies have revealed the dynamic, heterogeneous nature of bacterial IBs, which alongside of misfolded protein also contain protein species with amyloid structure as well as native-like and correctly folded protein (Garcia-Fruitos et al 2005;Park et al 2012;Jäger et al 2019a;Jäger et al 2018;Jäger et al 2019b;Kloss et al 2018a, b;Lamm et al 2020;Zhou et al 2012;Wang et al 2015;Jiang et al 2019;Wu et al 2011;Lin et al 2013;Diener et al 2016;Choi et al 2011;Nahalka and Nidetzky 2007;Nahalka et al 2008;Nahalka 2008;Nahalka and Patoprsty 2009;Koszagova et al 2018;Huang et al 2013;Arie et al 2006). Thus, more and more evidence suggests that those properties are to a certain degree an inherent feature of all IBs and that all cytoplasmic proteins exist in a conformational equilibrium between soluble-folded, partially misfolded, and insoluble aggregates.…”